Visualization of a protein-protein interaction at a single-molecule level by atomic force microscopy

Area of Science:

• Biophysics
• Nanotechnology
• Biochemistry

Background:

• Atomic force microscopy (AFM) offers high-resolution imaging of biological systems.
• In situ high-speed imaging advances biomolecule interaction monitoring.
• Existing methods face challenges in studying protein-protein interactions.

Purpose of the Study:

• To develop and validate an alternative ex situ AFM method for studying biomolecular interactions.
• To enable visualization of identical molecules before and after binding events.
• To investigate the complex formation between von Willebrand factor (VWF) and factor VIII (FVIII).

Main Methods:

• An ex situ AFM imaging approach using nanoscale scratches as markers for molecule relocation on mica.
• Imaging of identical molecules before and after reaction with binding partners.
• Studying VWF-FVIII complex formation, including specificity testing with high salt buffer.

Main Results:

• The method successfully visualized FVIII binding to VWF, characterized by appended globular domains.
• Protein reactivity was maintained after immobilization and intermittent drying.
• Specificity of VWF-FVIII interaction was confirmed under inhibitory conditions.

Conclusions:

• The developed ex situ AFM technique provides a new perspective for studying protein-protein interactions.
• This method overcomes limitations of in situ imaging and other ex situ approaches.
• Proteins retain functionality on solid substrates for subsequent interactions.